Friend spleen focus-forming virus (SFFV) is a potent pathogenic agent, which causes extensive proliferation of spleen cells shortly after infection, leading to the rapid development of erythroleukemia. Genetic evidence strongly implicates the env gene of this virus as the major pathogenic determinant of these viruses. The SFFV env gene is defective in comparison to its normal MuLV counterpart, and its gene products are not incorporated into viral particles and thus cannot be involved in viral replication. These facts make SFFV an ideal system for elucidating the functional roles of retroviral env genes in leukemogenesis. We have recently shown that the mature SFFV env gene product, gp65, is efficiently secreted from cells, and we have found that partially purified preparations of gp65 stimulate the proliferation of erythroid progenitor cells in in vitro cultures. These results suggest that gp65 may be the molecule responsible for the pathogenicity of these viruses. In order to test this hypothesis, we propose to extend our studies of gp65 in the following ways. We will purify gp65 to homogeneity and test the biological activities of the purified material. Further biochemical studies of gp65 will be performed, including amino- and carboxy-terminal sequencing and the complete characterization of the post- translational modifications of this molecule and identification of their sites of attachment. Site-specific mutations will be introduced into molecularly cloned genomes of SFFV and MuLVs by oligonucleotide-directed mutagenesis, in order to determine the functional roles of the SFFV-specific structural features in the biological activities of these proteins, and in the pathogenicity of these viruses. Monoclonal antibodies and site- specific antisera will be prepared against different domains of gp65. The active site of gp65 will be identified by examining the effects of such antibodies on the in vitro and in vivo biological activities of this molecule. The effect of the regulatory Fv-2 gene on the response of hematopoietic cells to gp65 will be determined as an additional test of the biological specificity of the proliferative activity of this molecule. Finally, gp65 binding assays will be developed, and cell surface receptors for gp65 isolated and characterized, in order to study the role of these molecules in viral leukemogenesis.